Application of andrographolide in the preparation of a pharmaceutical for treatment of inflammatory bowel disease, andrographolide enteric targeting micropellet, and method for preparation thereof

ABSTRACT

The present invention relates to an andrographolide enteric targeting micropellet and method for preparation thereof; furthermore, the present invention also relates to uses of andrographolide and andrographolide enteric targeting micropellets in the preparation of a pharmaceutical for treatment of inflammatory bowel disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/910,173, filed on Feb. 4, 2016, which is U.S. national phase filingof PCT/CN2014/083810, filed on Aug. 6, 2014, all of which claim priorityto Chinese Patent Application No. 20130338444.1, filed on Aug. 6, 2013,the entire contents of each of which are incorporated by referenceherein in their entireties.

FIELD OF THE INVENTION

Present invention relates to the field of medicine. More specifically,the invention relates to an andrographolide enteric targetingmicropellet and method for preparation thereof. Also, present inventionrelates to an application of andrographolide and andrographolide enterictargeting micropellet in the preparation of a medicine for treatment ofinflammatory bowel disease.

BACKGROUND OF THE INVENTION

Andrographolide (C₂₀H₃₀O₅) is the diterpene lactone compound extractedfrom the plant Andrographis Paniculata. It is one of the main componentsin the Andrographis Paniculata Nees (APN) which is crowned as thenatural antibiotic because of its effects of anti-pathogenicmicroorganism, antipyresis, anti-inflammation, improving body immune,protecting liver by normalizing functioning of the gallbladder andanti-tumor etc. The andrographolide belongs to the diterpene lactonecompound. Being an herbal extract, it has advantages of less sideeffects, better anti-inflammation and extensive source with acompetitive price.

Inflammatory bowel disease (IBD) is a recurrent chronic inflammatorydisease on intestinal, mainly including the ulcerative colitis (UC) andthe Crohn's disease (CD). Their definite cause and pathogenesis have notyet been eradicated and therefore there are insufficient effectivetreatment methods in clinic. The clinical manifestations of UC included:the intestinal injury, most of which firstly appeared in the distalcolon and sigmoid colon; mainly the left abdomen dull pain or sustainedsecret pain, which may be relieved after diarrhea: the myxoid-like andpus-blood-like stool accompanied with the tenesmus. The clinicalmanifestations of CD included: mostly the abdomen pain; the left abdomencolic pain or spastic sharp pain characterized by paroxysmal occurrenceand colic pain occurs post meal; the myxoid-like and watery stoolaccompanied with constipation alternative with diarrhea. A series ofdisease may be more likely to be found in CD than in UC, such as theintestinal stenosis, the intestinal obstruction, the intestinal fistula,the intestinal polyp and even the carcinogenesis.

Crohn's disease (CD) is identified as one of the IBD. Usually, thesymptoms of inflammation, congestion or swollen lymph may occur in thecolon, small intestine or stomach. The main difference from UC lies ininflammation position and the inflammation itself. The Crohn's diseasemay affect any segment of digestive system, e.g. the small intestine,colon, stomach and esophagus, which is common in terminal ileum andadjacent colon segment and right-half colon. UC, however, just occurs incolon and rectum, which is common in rectum and sigmoid.Microscopically, the Crohn's disease may affect whole inner wall ofbowel, while UC is restricted to mucosa.

Crohn's disease is a chronic and recurrent disease. Effectivelytherapeutical drugs have not yet been developed for its unknown cause.By now, drugs used for treating Crohn's disease mainly includeglucocorticoid, salicylic acid, immunosuppressive agents, antibiotics,methotrexate and biological agents (e.g. infliximab). Although thesedrugs are proven to be able to change the natural process of disease,they can not completely alleviate the conditions of disease and decreasethe incidence of complications. Moreover, the chemicals ofglucocorticoid and immunosuppressive agents often cause obvious adversereaction and longtime administration will likely result in damage to thebody. Hence, we need to develop a new medicine and its formulationthereof for treatment of the Crohn's disease.

On the other hand, the colon drug delivery has been regarded as adifficult issue in R&D for a long time, which is determined by colon'sown physiological characteristics. It is well-known that the colon islocated in the bottom half of the digestive tract, that drugs are verydifficult to reach the colon when administrated orally and that enemaadministration is both inconvenient and painful. As a result of this,the enteric targeting preparation technique emerged. Oral colon-specificdrug delivery system (OCSDDS) refers to a drug delivery technique, whichmakes the drug pass through top half of the digestive tract of thestomach and duodenum unchangedly. The medicine is not released untiltransferred to the ileum to demonstrate local or systematic therapeuticeffects. The common-used OCSDDS techniques are divided into thepH-dependent type and the enzyme degraded type.

The pH-dependent OCSDDS is to achieve the colon specific delivery byutilizing the different pH value of each part in human gastrointestinaltract. Usually, the gastric pH value of healthy people is lowest at 1˜3,the duodenum at 4˜6, the jejunum at 6˜7, ileum at 7˜7.5 and colon at7˜8.

Now, common-used enteric-coating materials have different pH points atwhich to dissolve. The first type began to dissolve at pH value ≧5.5,the second at ≧6.0 and the third at ≧7.0. Up to now, the drug is wrappedby using the third enteric polymer to coat in the pH-dependententeric-targeting preparation. It may be achieved that the drug does notrelease through the top half of the gastrointestinal tract untiltransferred to the ileum. All Chinese patents (CN1981743, CN101209246,CN103315959) are involved in this technique. As shown in clinicalstudies, however, gastrointestinal pH values among different individualsare far away from each other. There is a gap between IBD patients andhealthy people, and the colon pH value in colitis patients is lower thanhealthy people. As a result, when using this kind of polymer alone, thedrug will not be released in vitro and expelled with stool.

As for the andrographolide enzyme-degraded oral colon-specific delivery,the prior arts include steps: coating blank pellet with theandrographolide to have a drug-loading micropellet and wrapping saidmicropellet with monosaccharide pore-forming agents-inclusive insolublepolymer. Said polymer film does not release in the stomach and smallintestine until reaching the colon. The monosaccharide in the film isdegraded by colon enzymes. After the pore is formed, the drug isgradually dissolved and released. Although this technique has overcomedefects of difference among individuals in the pH-dependent OCSDDS,there are problems. Because the monosaccharide, e.g. the Guar gum isdissolved in water and the drug will be soon released from pores formedby dissolution of monosaccharide after entering the body, it isdifficult to ensure that effective amount of drug reaches the colon. Inaddition, the molecular of monosaccharide is structurally rigid. Oncebeing embedded into the polymer chain, it will not only affect theextensibility of polymer chain, but also destroy the integrity ofpolymer membrane, which will make the coating membrane crisped andeasily broken. Hence, the risk is increased that the membrane may beearly broken during transportation or by gastrointestinal peristalsis.As a result of this, developing new preparations of andrographolide isstill needed for treating the IBD.

CONTENT OF THE INVENTION

In the first place, the objective of present invention is to provide anew use of andrographolide, particular to a use of andrographolide inpreparation of medicine for treating IBD. Wherein, said IBD includes UCand Crohn's disease.

On the other hand, the objective of present invention is to provide anew pH-dependent enteric targeting preparation. That is to say, twotypes of pH-dependent polymers are jointly used to achieve the purposeof targeting release in vitro with different pH values. In particular,the present invention relates to an andrographolide enteric targetingmicropellet. Said micropellet is used for better treating IBD, forexample UC and Crohn's disease. Also, the present invention relates to amethod for preparing said pH-dependent enteric targeting preparation.

Preferably, present invention relates to the technical scheme, asfollows:

1. An andrographolide enteric targeting micropellet is composed of ablank pellet, a drug layer and an enteric coating layer, wherein saiddrug layer contains the andrographolide, the polymer A dissolved undercondition of pH≧7.0 and the excipient; said ratio of the andrographolideand polymer A is 1:2˜1:0.2 by weight; weight gain of the drug layer is20 wt %˜100 wt %, preferably 30 wt %˜80 wt %; said enteric coating layercontains the polymer B dissolved under condition of pH≧5.5 and theexcipient; weight gain of the enteric coating layer is 5 wt %˜30 wt %,preferably 8 wt %˜20 wt %, most preferably 10 wt %˜18 wt %.2. The micropellet according to 1^(st) paragraph, wherein said druglayer contains excipients of the plasticizer, anti-sticking agent,pigment, hydrophilic polymer and surfactant; said enteric coating layercontains excipients of the plasticizer and anti-sticking agent;preferably said excipients optionally include the hydrophilic polymerand pigment.3. The micropellet according to 1^(st) paragraph, wherein said polymer Ais the copolymer of methacrylic acid and methyl methacrylate and polymerB is the copolymer of methacrylic acid and ethyl acrylate.4. The micropellet according to 1^(st) paragraph, wherein said polymer Ais the copolymer of methacrylic acid and methyl methacrylate in a ratioof 1:2 and/or the polymer B the copolymer of methacrylic acid and ethylacrylate in a ratio of 1:1.5. The micropellet according to 1^(st) paragraph, wherein saidplasticizer is selected from one or more kinds of the triethyl citrate,the dibutyl sebacate, the propanediol and PEG, accounting for 10˜70 wt %of the polymer A, preferably 10˜20 wt %; said anti-sticking agent is thetalc, accounting for 25˜100 wt % of the polymer A, preferably 30˜50 wt%; or said anti-sticking agent is the glyceryl monostearate, accountingfor 2˜20 wt % of the polymer A, preferably 5˜10 wt %.6. The micropellet according to 1^(st) paragraph, wherein the diameterof said blank pellet is 200˜600 μm, preferably 300˜500 μm, accountingfor 10˜70 wt % of the formula, preferably 20˜60 wt %.7. The micropellet according to 2^(nd) paragraph, wherein saidingredients are present in proportion by weight parts: blankmicropellet:andrographolide:polymer A:plasticizer:anti-stickingagent:surfactant=200:(10-100):(10-100):(1-15):(1-30):(0-3), preferably200:(15-66):(13-74):(2-13.5):(3-27):(0-1.32), most preferably200:(20-50):(30-60):(5-10):(5-20):(0.5-1.2).8. The preparation method according to any one micropellet of1^(st)˜7^(th) paragraphs comprising following steps:(1) applying drug to the blank pelleta). dispersing the polymer A into a pharmaceutical solvent to let themdissolve fully by mechanical stirring; adding the excipient into thepolymer A solution and then adding the andrographolide to have thepolymer A coating solution by well stirring;b). weighing the blank pellet and charging into a fluidized bed;adjusting air flow to such a degree that the micropellet is wellfluidized; opening the heating device and until temperature of thematerial reaches preset value, the peristaltic pump is started to makethe polymer A coating solution atomized through a spray gun to obtain adrug-loading micropellet by dispersing on the surface of said blankpellet;(2) preparation of the enteric coating layera). dispersing the polymer B into a pharmaceutical solvent to let themdissolve fully by mechanical stirring; adding the excipient into thepolymer B solution to have the polymer B coating solution by wellstirring;b). charging aforesaid drug-loading micropellets into a bottom-spraydevice of the fluidized bed, and the polymer B coating solution isuniformly spread to form the enteric coating layer; the weight gain is 5wt %˜30 wt %.9. The preparation method according to 8^(th) paragraph comprisingfollowing steps:(1) applying drug to the blank pelleta). dispersing the polymer A into a pharmaceutical ethanol to make thecontent of polymer A at 5 wt %; fully dissolving by mechanical stirringand continuing to stir uniformly; adding the excipient of theplasticizer, anti-sticking agent and the surfactant of sodium dodecylsulfate into the polymer A solution and then adding the andrographolideto have the polymer A coating solution by well stirring;b). weighing the blank sucrose pellet in a diameter of 200˜600 μm andcharging into a fluidized bed; adjusting air flow to such a degree thatthe micropellet is well fluidized; opening the heating device to keepthe temperature of the material at 25˜35° C. and until the temperaturereaches preset value, the peristaltic pump is started to make thepolymer A coating solution atomized through a spray gun to obtain adrug-loading micropellet by dispersing on the surface of said blankpellet;(2) preparation of the enteric coating layera). dispersing the polymer B into the pharmaceutical ethanol to let themdissolve fully by high-speed shearing mechanical stirring; adding theexcipient of the plasticizer and anti-sticking agent into the polymer Bsolution to have the polymer B coating solution by well stirring;b). charging aforesaid drug-loading micropellets into a bottom-spraydevice of the fluidized bed, and the polymer B coating solution isuniformly spread to form the enteric coating layer; the weight gain is 8wt %˜20 wt %.10. An andrographolide enteric targeting preparation, characterized inthat any one micropellet of 1^(st)˜7^(th) paragraphs is prepared intogranule or capsule.11. Use of andrographolide, any one micropellet of 1^(st)˜7^(th)paragraphs or the targeting preparation of 10^(th) paragraph inpreparation of medicine for treating the IBD.12. The use according to 11^(th) paragraph, wherein said IBD is UC orCrohn's disease.13. The use according to any one of 11^(th) or 12^(th) paragraphincluding improving colon adhersion, intestinal wall red swelling andthickening and decreased elasticity.14. The use according to any one of 11^(th) or 12^(th) paragraphincluding reducing colon ulcer surface, hemorrhagic spot and poferation.15. The use of any one of 11^(th)˜14^(th) paragraphs, characterized inthat said medicine is prepared into an enteric-coated preparation.16. The use of any one of 11^(th)˜14^(th) paragraphs, characterized inthat said medicine is prepared into an enteric targeting micropellet.17. The use of 16^(th) paragraph, characterized in that said enterictargeting micropellet is prepared into a granule or capsule.

DESCRIPTION OF THE DRAWINGS

FIG. 1 indicated the effect of the andrographolide on the body weight ofTNBS-caused cotilis mouse.

FIG. 2 was the general observation pictures of each group in TNBS-causedUC rats (A-F).

FIG. 3 showed the zebra fish intestine in tested drug group, Crohn'sdisease model group and blank control group (The region shown by greendotted line was the zebra fish intestine).

FIG. 4 indicated the improvement degree of bowel dilatation in Crohn'sdisease model group after being administrated with the andrographolideat different doses (compared with the blank control group).

FIG. 5 indicated the therapeutical ratio of andrographolide at differentdoses in Crohn's disease model group calculated in accordance with thebowel area.

FIG. 6 showed the distribution of neutrophil in zebra fish intestinaltissue after being treated with drugs. (The region shown by yellowdotted green line was the zebra fish intestine and the one by greenbright spot was the neutrophil.)

FIG. 7 was the cumulative release amount of drug in the phosphate bufferof simulated intestinal fluid (pH 6.5).

FIG. 8 was the cumulative release amount of drug in the phosphate bufferof simulated intestinal fluid (pH 7.2).

DETAILED DESCRIPTION OF THE INVENTION

In first embodiment of this invention, said IBD chiefly relates to UCand Crohn's disease. By studying, the inventor of present inventionconcluded: the andrographolide had an improving effect on the colonadhersion, intestinal red swelling and thickening and decreasedelasticity. In addition, the andrographolide was able to reduce colonulcer surface, hemorrhagic spot and poferation.

In one embodiment of present invention, said medicine of presentinvention includes any one qualified drug prepared by using theandrographolide as an active pharmaceutical ingredient (API).Preferably, said medicine of present invention refers to apharmaceutical composition comprising the andrographolide alone or incombination with other ingredients.

In one embodiment of present invention, the andrographolide belongs toprior arts, which is either commercially available or prepared by aconventional method. For example, the andrographolide is prepared by thefollowing method: the leaves of Andrographis paniculata is soaked in 95%(v/v) ethanol and the resulting ethanol liquid is decolored with theactivated carbon and the ethanol is recovered by distillation to give aconcentrated liquid. The liquid is allowed to stand still to have coarsecrystal. Said coarse crystal is added with 15 times (15×) 95% (v/v)ethanol, dissolved by heating, decolored with activated carbon andfiltered immediately. The liquid is allowed to stand still to give alight-yellow crystal by recrystallization. The obtained crystal isrefined by washing with distilled water, chloroform and menthol to havethe final product of andrographolide.

In one embodiment of present invention, the andrographolide is desirablyadministrated in a form of pharmaceutical composition. Said compositionmay be conventionally used in combination with one or more kinds ofphysiologically acceptable excipients or carriers. If possible, theandrographolide acts as APT and administrated directly to patients,preferably the API is administrated directly as a preparation. In termsof integration with other ingredients and safety on the subject, saidexcipient must be accepted pharmaceutically.

In one embodiment of present invention, said pharmaceutical compositioncan be prepared into any one of pharmaceutically acceptable dosage formswhen applied clinically, including but not limited to: the tablet, e.g.the sugar-coated tablet, film-coated tablet and enteric-coated tablets;capsule, e,g, the hard capsule, soft capsule or enteric-coated capsule;injection; suppository, e.g. the intestinal suppository; drop etc,preferably the enteric-coated suppository, e.g. the enteric-coatedtablet and enteric-coated capsule.

In one embodiment of present invention, the pharmaceutically acceptableexcipients may be added when preparing said pharmaceutical composition.

In one embodiment of present invention, oral preparations may includeconventional excipients, e.g. the adhesive, filling agent, diluent,tableting agent, lubricant, disintegrating agent, colorant agent,flavoring agent, wetting agent. If necessary, the tablet may be coated.Said filling agents include cellulose, mannitol, lactose and otheranalogous filling agent. Competent disintegrating agents include starch,polyvinylpyrrolidone (PVP) and starch derivative (e.g. sodiumhydroxyethyl starch). Competent lubricants include magnesium stearate.Competent pharmaceutically acceptable wetting agents include sodiumdodecyl sulfate. The oral solid preparations can be prepared by aconventional method of blending, filling, tabletting or granulating etc.Repeated blending is performed to make the API distributed uniformly incompositions in which lots of filling agents are used.

In one embodiment of present invention, as for injections, saidpreparation unit contains the andrographolide and aseptic excipients.Whether said API is dissolved or suspended in the liquid depends on thetype and concentration of excipients. Generally, solution is prepared bydissolving the TCM formulation in the excipients as the API,sterilizing, loading into an appropriate vial or ampoule and sealing.Some pharmaceutically acceptable adjuvant, e.g. local anaesthetic,preservative and buffering agent can be added as required. In order toimproving its stability, before loaded into the vial, this TCMformulation of present invention can be frozen and treated in vacuum toremove water.

In one embodiment of present invention, the effective daily dose of themedicine for adult treatment is always in the range of 0.02˜5000 mg whenused for prevention and treatment of UC and Crohn's disease, preferably1˜1500 mg. Said dose needed for treatment is either a single dose or amultidose, at which the medicine is administrated at proper intervals,such as twice, triple, four times or more per day. The preparation ofpresent invention may include 0.1 wt %˜99.9 wt % of the API.

In second embodiment of this invention, a new pH-dependent enterictargeting preparation is provided, which is characterized that saidandrographolide enteric targeting micropellet is composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer contains the andrographolide, the polymer A dissolved undercondition of pH8≧7.0 and the excipient; said ratio of theandrographolide and polymer A is 1:2˜1:0.2 by weight; weight gain of thedrug layer is 20 wt %/˜100 wt %, preferably 30 wt %˜80 wt %; saidenteric coating layer contains the polymer B dissolved under conditionof pH≧5.5 and the excipient; weight gain of the enteric coating layer is5 wt %˜30 wt %, preferably 8 wt %˜20 wt %, most preferably 10 wt %˜18 wt%.

Among these, said polymer A is the copolymer of methacrylic acid andmethyl methacrylate, preferably the copolymer of methacrylic acid andmethyl methacrylate in a ratio of 1:2 and polymer B is the copolymer ofmethacrylic acid and ethyl acrylate, preferably the copolymer ofmethacrylic acid and ethyl acrylate in a ratio of 1:1.

In a preferred embodiment of present invention, the polymer A isselected from the Eudragit S100 purchased from Rohm Inc, and the polymerB is the Eudragit L series polymers, most preferably the EudragitL100-55.

In one embodiment of present invention, said drug layer containsexcipients of the plasticizer, anti-sticking agent, pigment, hydrophilicpolymer and surfactant. Preferably, the surfactant is selected from thesodium dodecyl sulfate (SDS) or Tween-80 with adding amount of 0˜5 wt %of the andrographolide, preferably 1 wt %˜3 wt %.

In one embodiment of present invention, said plasticizer is selectedfrom one or more kinds of the triethyl citrate, the dibutyl sebacate,the propanediol and PEG, accounting for 10˜70 wt % of the polymer A,preferably 10˜20 wt %; said anti-sticking agent is the talc, accountingfor 25˜100 wt % of the polymer A, preferably 30˜50 wt %; or saidanti-sticking agent is the glyceryl monostearate, accounting for 2˜20 wt% of the polymer A, preferably 5˜10 wt %.

In one embodiment of present invention, the diameter of said blankpellet is 200˜600 μm, preferably 300˜500 μm, accounting for 10˜70 wt %of the recipe quantity, preferably 20˜60 wt %. Said blank pellet is theconventional pharmaceutical pellet, preferably the blank sucrose pelletor microcrystalline cellulose pellet.

In one embodiment of present invention, said ingredients are present inproportion by weight parts: blank pellet:andrographolide:polymerA:plasticizer:anti-stickingagent:surfactant=200:(10-100):(10-100):(1-15):(1-30):(0-3).

Preferably, said ingredients are present in proportion by weight parts:blank micorpellet:andrographolide:polymer A:plasticizer:anti-stickingagent:surfactant=200:(15-66):(13-74):(2-13.5):(3-27):(0-1.32).

Most preferably, said ingredients are present in proportion by weightparts:blank micorpellet:andrographolide:polymer A:plasticizeranti-stickingagent:surfactant=200:(20-50):(30-60):(5-10):(5-20):(0.5-1.2).

Wherein, excipients in the enteric coating layer include the plasticizerand anti-sticking agent and are selected as depicted before. Theplasticizer accounts for 15 wt % of the polymer B and the anti-stickingagent 30 wt %.

Optimized 7 formulas of the blank micropellet and drug layer are presentas follows:

Blank micropellet andrographolide Eurdragit S anti-sticking SurfactantNo. (g) (g) (g) Plasticizer (g) agent (g) (g) 1 200 50 15 2.1 4.5 1.00 2200 66 20 3 6 1.32 3 200 66 22 3.3 6.6 1.32 4 200 44 13 2 3 0 5 200 5818 3.6 5.4 1.16 6 200 15.2 40 6 12 0.4 7 200 52 74 13.5 27 1.26

The preparation method for preparing the andrographolide enterictargeting micropellet is present as follows:

(1) applying drug to the blank pelleta). dispersing the polymer A into a pharmaceutical solvent to let themdissolve fully by mechanical stirring; adding the excipient into thepolymer A solution and then adding the andrographolide to have thepolymer A coating solution by well stirring;b). weighing the blank pellet and charging into a fluidized bed;adjusting air flow to such a degree that the micropellet is wellfluidized; opening the heating device and until temperature of thematerial reaches preset value, the peristaltic pump is started to makethe polymer A coating solution atomized through a spray gun to obtain adrug-loading micropellet by dispersing on the surface of said blankpellet;(2) preparation of the enteric coating layera). dispersing the polymer B into a pharmaceutical solvent to let themdissolve fully by high-speed shearing mechanical stirring; adding theexcipient into the polymer B solution to have the polymer B coatingsolution by well stirring;b). charging aforesaid drug-loading micropellets into a bottom-spraydevice of the fluidized bed, and the polymer B coating solution isuniformly spread to form the enteric coating layer; the weight gain is 5wt %˜30 wt %.

Preferably, the preparation method for preparing the andrographolideenteric targeting micropellet is present:

(1) applying drug to the blank pelleta). dispersing the polymer A into a pharmaceutical ethanol to make thecontent of polymer A at 5 wt %; fully dissolving by high-speed shearingmechanical stirring and continuing to stir uniformly; adding theexcipient of the plasticizer, anti-sticking agent and the surfactant ofsodium dodecyl sulfate into the polymer A solution and then adding theandrographolide to have the polymer A coating solution by well stirring;b). weighing the blank sucrose pellet in a diameter of 200˜600 μm andcharging into a fluidized bed; adjusting air flow to such a degree thatthe micropellet is well fluidized; opening the heating device to keepthe temperature of the material at 25˜35° C. and until the temperaturereaches preset value, the peristaltic pump is started to make thepolymer A coating solution atomized through a spray gun to obtain adrug-loading micropellet by dispersing on the surface of said blankpellet;(2) preparation of the enteric coating layera). dispersing the polymer B into the pharmaceutical ethanol to let themdissolve fully by high-speed shearing mechanical stirring; adding theexcipient of the plasticizer and anti-sticking agent into the polymer Bsolution to have the polymer B coating solution by well stirring;b). charging aforesaid drug-loading micropellets into a bottom-spraydevice of the fluidized bed, and the polymer B coating solution isuniformly spread to form the enteric coating layer; the weight gain is 8wt %˜20 wt %.Besides, present invention is involved in an andrographolide enterictargeting preparation and said preparations include the capsule orgranule prepared from aforesaid micropellets by a conventional method.

Advantages

A specific pH-dependent technique has been used, namely the joint use oftwo pH-dependent polymers, to make it enteric targeting release inbodies of different colon pH values.

{circle around (1)} The first type of enteric coating material, e.g.Eudragit L 100-55, has been used in the enteric coating layer, ensuringthat the medicine does not release in the stomach until exposing thedrug layer by quick dissolution after reaching the duodenum. {circlearound (2)} The second type of coating material (e.g. the Eudragit S100), as the middle layer, is used as a skeleton in the drug layer,among which the drug is uniformly distributed. Gradually, the drug isreleased by dissolution of enteric coating layer when the micropelletreaches the duodenum. Under low pH condition, however, the drug isreleased a little; only when approaching the end of small intestine atpH close to 7, the drug is released quickly, because the Eudragit S 100in the drug layer has a retarding effect in the low pH condition.

As depicted before, the andrographolide enteric targeting micropellet ofpresent invention has a three-layer structure: the blank pellet, thedrug layer and the enteric coating layer. The enteric coating layer iskept intact at pH below 5.5 when the preparation goes into the stomach.After reaching the duodenum, however, the enteric coating layer burstsinto dissolving to expose the drug layer. The Eudragit S 100 in the druglayer plays a dual role of sustained release and enteric dissolution.The andrographolide is uniformly distributed in the Eudragit S 100.After reaching the duodenum, the outside layer is dissolved. As soon asexposing to the body fluid, the andrographolide starts to release.Because the pH value on this position is low, the limited amount ofEudragit S 100 is dissolved and very few amount drugs released at veryslow rate. With transfer of drug to low half of gastrointestinal tract,the pH value goes up gradually, the dissolution rate of Eudragit S 100is accelerated and the release rate of drug increased. As a result, mostof the drug is not released until approaching ileum and colon, capableof treating intestinal inflammation.

The diameter of said blank pellet of present invention is 200˜600 μm,much less than the clinical commonly-used blank micropellet (500˜1000μm). This will not only help improving the specific surface area, butalso the contact area between the drug and the inflammatory site,ensuring that the andrographolide plays the therapeutical effect on theIBD. The blank pellet accounts for 10 wt %˜70 wt % of the formulaquantity.

The Eudragit series polymers are used for coating as a film material incombination with the plasticizer and anti-sticking agent added in theformula. Wherein, the purpose of using plasticizer is for not onlyreducing glass transition temperature and minimum film formingtemperature (MFFT), but also increasing flexibility of the polymer film.The purpose of using anti-sticking agent is for preventing the film frombeing sticky, causing bonding mutually among the blank pellets. Thesurfactant may be used for increasing the wetting effect on the drug.

The pharmacological effects of andrographolide and its new preparationare proven by following experiments.

Pharmacological Research 1 Pharmacological Study of Andrographolide onTNBS-Induced UC 1. Aim

The mice IBD model was used to perform a preliminary evaluation of theandrographolide for treating UC and Crohn's disease

2 Materials 2.1 Animals

50 SPF Balbc/c male mice, weighing 18˜24 g, were provided from BeijingWeitonglihua Experimental Animals Inc and Certificate No. 2011-0012

2.2 Raising conditions

Animals were raised in a barrier animal room, 10 mice in each cage, withtemperature at 20˜25° C. and relative humidity at 40˜60% free access towater and padding materials replaced daily.

2.3 Tested medicine and reagentsTested medicine: andrographolide, white dry powder, was provided byTasly Modern TCM Resource Inc with yield rate of 98% and purity of 98%.Positive drug: Sulfasalazine was purchased from Shanghai SanweiPharmaceutical Inc with the batch No 200206C11 and specification: 250mg/tablet, 12 tablet×5.Reagents: TNBS was purchased from Sigma Inc with the batch No 033K5020and specification: 5% (w/v) and 10 ml/bottle.

3 Method

3.1 Preparation of model

5% (w/v) TNBS solution was diluted with double distilled water and mixedwith 50% (v/v) ethanol in equivalent volume to have 1.5% (w/v) TNBSsolution. In the model group, the mice were anesthetized with 1 wt %pentobarbital sodium at dose of 0.05 ml/10 g body weight. After beinganesthetized, the mice were administrated with 1.5% (w/v) TNBS solutionat dose of 0.05 ml/mouse by gently inserting stomach perfusion device toabout 3 cm depth of colon via anus and the IBD was induced. In thesaline group, the solution was injected into the colon at dose of 0.1 mlmouse. In the normal group, 50% (v/v) ethanol was injected into thecolon at 0.1 ml mouse.

3.2 Grouping and administration

After one week adaptive feed, all animals were randomly divided into 5groups according to body weight, 10 mice in one group: the normalcontrol group, the model group, the andrographolide low-dose group (20mg/kg/d), the andrographolide high-dose group (40 mg/kg/d) and thepositive control group (Sulfasalazine, 300 mg/kg/d). 2 hours aftermaking model, the andrographolide was administrated orally twice dailyin each treating groups and once daily in the positive control group.After successive administration for 7 days, the abdomen was opened 24hours after last administration to observe adhesion between colon andother organs. The colon was separated and weighed.

3.4 Evaluation indices1. Body weight: after making model, body weight was measured every dayto observe the change.2. Evaluation of inflammation: the abdomen was opened 24 hours afterlast administration to observe adhesion between colon and other organs.Every segment of colon was taken out and weighed to calculate the ratioof colon weight to body weight as the colon index. The formula waspresent as follows:

Decrease rate of colon specific weight=(colon index of model group-colonindex of treatment group)/colon index of model group×100%

3. Histopathological examination:0 score, no inflammatory symptoms1 score, low-grade inflammation with no structure change2 score, low-grade leukocyte infiltration3 score, high-grade leukocyte infiltration, high vascular density, cryptextension, thickened colon wall and superficial ulcer4 score, high-grade leukocyte infiltrating to mucus layer, cryptextension, decrease of goblet cell, high vascular density, thickenedcolon wall and extensive ulcer

3.5 Statistics

SPSS11.5 software was used for analysis and data were expressed as x±S.Variance analysis was used for comparison of significant differenceamong groups. P<0.05 showed a statistically significant difference.

4. Results

4.1 Effect of the andrographolide on the BW of TNBS-induced IUC mice

The body weight index was able to generally reflect the overall healthstatus of mice. Compared with the normal control group, the mouse growthwas affected after forming UC by rectal administration of TNBS: slowgrowth of body weight. Being treated with the andrographolide or thesulfasalazine, the body weight grew faster than the model control group.Data were seen in FIG. 1.

4.2 Effect of the colon weight, colon indices and rate of colon specificweight

After UC was indiced by TNBS, the colon weight and colon indices wereobviously higher than the normal control group, illustrating thesuccessful modeling. After 7 days of treatment with drugs, compared withthe model group, the colon weight in the andrographolide high-dose groupand the positive control group was reduced significantly (P<0.01); thecolon indices in the andrographolide high-dose group, low-dose group andthe positive control group was reduced significantly (P<0.05, P<0.01).The decrease rates of colon specific weight were 33.20%, 58.96% and47.87. Data were seen in Table 1.

TABLE 1 effect of the andrographolide on colon weight, colon indices andthe decrease rate of colon specific weight in the UC mice Colon weightBody Decrease rate of colon (g) weight (g) Colon indices specific weight(%) Normal control 0.17 ± 0.03 22.92 ± 1.01 0.77 ± 0.10 — group Modelgroup 0.55 ± 0.15 16.32 ± 1.06 3.35 ± 0.88 — Andrographolide 0.43 ± 0.1219.95 ± 2.05 2.24 ± 0.90* 33.20 low-dose group Andrographolide 0.29 ±0.09** 20.95 ± 1.09 1.38 ± 0.46** 58.96 high-dose group Positive control0.33 ± 0.06** 19.13 ± 0.99 1.75 ± 0.43** 47.87 group Compared with themodel group: *P < 0.05: **P < 0.014.3 Effect of the andrographolide on histopathological changes of colonin TNBS-induced UC mice

After UC was induced by TNBS, a series of symptoms occurred: colonadhesion, intestinal wall red swelling and thickening, decreasedelasticity, UC surface, colon hemorrhagic spot and poferation,indicating extensive inflammatory injuries in colon. The intestinalelasticity in andrographolide and positive control groups was higherthan that in the model group and the colon weight much less than themodel group, indicating that the inflammatory reaction of colon adhesionand exudation was weaker than the model group. Histopathologicalinjuries of intestinal mucosa and wall caused by Ulcer and hemorrhagewere examined with naked eyes; the colon histopathological injuries inthe andrographolide high-dose group and the positive control group weremuch lower than that in the model group. Data were seen in Table 2.

TABLE 2 effect of andrographolide on the colon score in TNBS-induced UCmice Colon score Normal control group 1 Model group 3.7 Andrographolidelow-dose 3.2 group Andrographolide 1.8** high-dose group Positivecontrol group 2.8* Compared with the model group: *P < 0.05: **P < 0.01

5 Conclusions

There were a lot of methods to build animal colitis model, TNBS/ethanolinduced model was the most similar with the UC pathological changes inclinic. Ethanol destroyed intestinal mucosal barriers and TNBS, as ahapten, would make T lymphocytes sensitized by combining with tissueproteins to cause the intestinal inflammation after inducing autoimmunereaction. The model rats caused by this method have lots of similaritieswith clinical symptoms of US patients: the stool change and intestinalgeneral morphological and histological change.

In this study, the UC model mice were used to preliminarily evaluate thetreating effect of andrographolide. As shown in the results, thehigh-dose andrographolide (40 mg/kg/d) was able to delay the descendingtrend of body weight in mice of model group, which, compared with themodel group, could significantly reduce the colon indices (P<0.01),ameliorate colon pathological changes and the decrease rate of colonspecific weight was 58.96%. Considering aforesaid indices, theandrographolide had a significantly improving effect on the UC in mice,having a certain therapeutical effect on UC and also capable of treatingCrohn's disease.

Pharmacological Research 2 Study on Therapeutical Effect ofAndrographolide on DSS-Induced UC

1. Aim

Efficacy of andrographolide on DSS-induced UC was evaluated.

2. Animals

84 SPF Balbc/c male mice, weighing 18˜22 g, were provided from GuangdongMedical Experimental Animal Center and Certificate number SYXK2008-0002. Raising conditions: 7 mice per cage raised in a group;temperature and humidity: 20-26° C. and 40-70%. The animals were lighted10 h/14 h days and nights intermittently. The condition of raising roomwas always remained unchanged, ensuring reliability of experimentresults. Animals were fed with the complete pellet feed (provided byGuangdong Medical Experimental Animal Center), free access to water viadrinking bottle.

3. Apparatuses and reagents

3.1 Apparatuses

3.1.1 Electronic balance, accuracy: 0.001 g, Zhongshan HengxinElectronics Inc.3.1.2 Auto dehydrating machine for organic tissue (TS-12N, XiaoganHongye Medical Device Inc.)3.1.3 Embedding machine for organic tissue (BM-VII, Xiaogan HongyeMedical Device Inc.)3.1.4 Machine for paraffin section (RM2135, Leica Inc. German)3.1.5 Machine for spreading and roasting section (CS-V, Xiaogan HongyeMedical Device Inc.)3.1.6 Auto staining machine for organic tissue (RS-18II, Xiaogan HongyeMedical Device Inc.)

3.2 Reagents

3.2.1 Andrographolide was provided by Tasly Modern TCM Resource Inc ofbatch number: 20140508 with purity>95%;3.2.2 Positive drug: mesalazine enteric-coated tablet was purchased fromJiamusi Luling Pharmaceutical Inc, Kuihua Pharmaceutical Group withbatch number: 140225.3.2.3 DDS (dextran sulfate sodium) was purchased from MPBIO Inc.4 Dose planning and grouping4.1 Grouping: quarantine-qualified 84 mice were randomly divided into 6groups: the model group, the positive drug group, the tested druglow-dose intragastric group, the tested drug high-dose intragastricgroup, the tested drug low-dose intracolonical group and the tested drughigh-dose intracolonical group, 14 mice per group. All animals drank 5%DDS solution daily for consecutive 14 days to establish UC model.Animals were administrated with the drug for treatment 2 days aftergiven 5% DDS drinking water.4.2 Dose: in this study dose of administration of mice in all groups wasdesigned on the basis of client's requirement and the same doses wereadopted in both intragastric and intracolonical groups. Animals were nottreated in the model group, mesalazine enteric-coated tablet (227.5mg·kg⁻¹·d⁻¹) in the positive drug group, andrographolide (60 mg·kg⁻·d⁻¹)in the low-dose intragastric group, andrographolide (180 mg·kg⁻¹·d⁻¹) inthe high-dose intragastric group, andrographolide (60 mg·kg⁻¹·d⁻¹) inthe low-dose intracolonical group and andrographolide (180 mg·kg⁻¹·d⁻¹)in the high-dose intracolonical group.

5 Method

5.1 Method of making UC model: all animals drank 5% DSS solution dailyfor consecutive 14 days.5.2 Method of administration: 2 days after modeling, mice were treatedintragastrically or intracolonically with drug at 1 mL/100 g once a dayfor consecutive 14 days.5.3 Method for sampling in experiment: 2 hours after lastadministration, mice were killed by cervical vertebra dislocation andabdomen was opened to separate the colon. Along mesenteric side, thecavity scissored off washed with 4° C. normal saline and spread on aplastic plate.5.4 Method for evaluating disease active index (DAI): the disease activeindex was assessed at 7^(th) and 14^(th) day after administration. Themethod was present together with following three parameters: the BW losspercentages (unchanged BW: 0 score, 1˜5: 2 score, 5˜10: 2 score, 10˜15:3 score, more than 15: 4 score), stool viscosity (normal stool: 0 score,loosen stool: 2 score, diarrhea: 4 score) and bloody stool (normalstool: 0 score, occult blood stool: 2 score and positive blood stool: 4score). Total score of three parameters was divided by 3 to give theDAI, namely DAI=(BW index+stool character score+stool blood score)/3.6 Observation indices6.1 Observation: the daily general clinical symptoms in mice wereobserved from beginning to end of the experiment in mice and the stool,mental status and death status were recorded.6.2 Body weight: BW was recorded weekly from beginning to end of theexperiment6.3 Calculation of DAI: DAI was calculated at 7^(th) and 14^(th) dayafter administration so as to assess disease activity.6.4 The colon mucosal tissue 8˜10 cm away from the anus was sampled,embedded with paraffin and stained with routine HE. Colon musco injurywas observed by microscope and histological injury was scored: ulcerinflammatory granuloma, fibrosis and pathological degree.

7. Statistics

Data were expressed as x±S. All numerical variables were analyzed withSPSSI1.5 software by one-way anava. T-test analysis was used forinter-group comparison. P<0.05 showed a statistically significantdifference.

8 Results

7 days after modeling, loosen stool, diarrhea, bloody stool and BW losswere observed in mice of model group, while the stool was more formed inthe treatment groups and occurrence of blood stool was slightly lessthan the model group. None of mice was found death during theexperiment.

8.1 Effect on the DAI

By comparing disease activity in all groups, diarrhea occurred in theDSS group 3^(rd)˜5^(th) days after drinking DSS and the occult bloodstool was positive. On 5^(th)˜7th days, different levels of gross bloodstool occurred. As such, diarrhea and occult blood stool occurred on3^(rd)˜5^(th) days in the positive control group and the andrographolidehigh-dose intracolonical group, but no obvious gross blood stool wasfound in mice after 5^(th) day. 14 days after administration, the numberof bloody stool and diarrhea in the positive control group andandrographolide high-dose intracolonical group was less than the modelgroup and DAI was significantly reduced (P<0.01) in comparison with themodel group and there was a certain decrease tendency in the number ofbloody stool and diarrhea in the positive control group andandrographolide high-dose intragastric group, having no statisticallysignificant difference in comparison with the model group. Data wereseen in Table 3.

TABLE 3 DAI score in all groups (x ± SD) Dose DAI Groups (mg/kg) 7^(th)day 14^(th) day Model group 1.63 ± 0.69 2.27 ± 0.91 Positive controlgroup 227.5 1.31 ± 0.14 1.10 ± 0.55** Andrographolide low-dose 60 1.63 ±0.99 2.01 ± 0.80 intragastric group Andrographolide high-dose 180 1.43 ±0.38 1.64 ± 0.83 intragastric group Andrographolide low-dose 60 1.67 ±0.65 1.77 ± 0.95 intracolonic group Andrographolide high-dose 180 1.36 ±0.64 1.35 ± 0.18** intracolonic group Compared with the model group: *P< 0.05, **P < 0.01

9. Conclusions

Up to now, mechanism of using DSS-induced model has not yet beenentirely clear. Perhaps, it is associated with many aspects: macrophagedysfunction; intestinal flora imbalance; influence of DNA synthesis ofcolonic epithelium cell by DSS negative charge; inhabitation toepithelial cell proliferation and destroying mucosal barrier, whichindicated a more ideal model for studying human IBD. As shown inresults, incidence of gross blood stool was reduced just 7 days afteradministration in the andrographolide high-dose intracolonic group (180mg/kg) and bloody stool and diarrhea ameliorated 14 days afteradministration. DAI score was significantly lower than the model group,which would be proven to have a certain protective effect on UC.

Pharmacological Research 3 Pharmacological Study of Andrographolide onTNBS-Induced UC 1. Aim

Efficacy of andrographolide on DSS-induced UC was evaluated.

2 Animals

84 SD rats of both sexes, weighing 180˜220 g, were provided fromGuangdong Medical Experimental Animal Center and Certificate number SYXK2008-0002. Raising conditions: 5 rats per cage raised in a group;temperature and humidity: 20-26° C. and 40-70%. The animals were lighted10 h/14 h days and nights intermittently. The condition of raising roomwas always remained unchanged, ensuring reliability of experimentresults. Animals were fed with the complete pellet feed (provided byGuangdong Medical Experimental Animal Center), free access to water viadrinking bottle.

3. Apparatuses and reagents

3.1 Apparatuses

3.1.1 Electronic balance, accuracy: 0.001 g, Zhongshan HengxinElectronics Inc.3.1.2 Auto dehydrating machine for organic tissue (TS-12N, XiaoganHongye Medical Device Inc.)3.1.3 Embedding machine for organic tissue (BM-VII, Xiaogan HongyeMedical Device Inc.)3.1.4 Machine for paraffin section (RM2135, Leica Inc. German)3.1.5 Machine for spreading and roasting section (CS-V, Xiaogan HongyeMedical Device Inc.)3.1.6 Auto staining machine for organic tissue (RS-18II, Xiaogan HongyeMedical Device Inc.)

3.2 Reagents

3.2.1 Andrographolide was provided by Tasly Modern TCM Resource Inc ofbatch number: 20140508 with purity>95%;3.2.2 Positive drug: mesalazine enteric-coated tablet was purchased fromJiamusi Luling Pharmaceutical Inc, Sunflower Pharmaceutical Group withbatch number: 140225.3.2.3 TNBS was purchased from SIGMA Inc with batch number: SLBG2566V.4 Dose planning and grouping4.1 Grouping: quarantine-qualified 84 rats were all used for making UCmodel. The successful rats were randomly divided into 6 groups: themodel group, the positive drug group, the tested drug low-doseintragastric group, the tested drug high-dose intragastric group, thetested drug low-dose intracolonical group and the tested drug high-doseintracolonical group, 14 rats per group.4.2 Dose: the same doses were adopted in both intragastric andintracolonical groups. Animals were not treated in the model group,mesalazine enteric-coated tablet (420 mg·kg⁻¹·d⁻¹) in the positive druggroup, andrographolide (30 mg·kg⁻¹·d⁻¹) in the low-dose intragastricgroup, andrographolide (90 mg·kg⁻¹·d⁻¹) in the high-dose intragastricgroup, andrographolide (30 mg·kg⁻¹·d⁻¹) in the low-dose intracolonicalgroup and andrographolide (90 mg·kg⁻¹·d⁻¹) in the high-doseintracolonical group.

5. Method

5.1 Method of making UC model: the rats were anesthetized. 2 mm-diameterlatex tube was gently inserted into the position about 8 cm inside ratsthrough anus and 50% ethanol TNBS solution (TNBS 125 mg/kg) was injectedinto intestinal cavity with an injector one time, 0.5 ml/rat. The tailof rats was lifted up for 30 s upended to make the model making agentfully infiltrate into the intestinal cavity of rats.5.2 Method of administration: the same dose (1 mL/100 g) was adopted inboth intragastric and intracolonical groups, once daily for consecutive5 days.5.3 Method for sampling in experiment: 2 hours after lastadministration, mice were killed by cervical vertebra dislocation andabdomen was opened to separate the colon. Along mesenteric side, thecavity scissored off washed with 4° C. normal saline and spread on aplastic plate.5.4 Scoring method for colon gross morphological injury was referred toScoring method for Mucosal Injury (Bjelkengren G, Aronsen K F,Augustsson N E, etc. Radioprotective effect of local administration oflysine vasopressin and triglycyl lysine vasopressin on the rectal mucosain rats [J]. Acta Oncol, 1995, 34(4):487-92) to record the mucosalinjury score: dot splinter blooding and small erythema (<1 mm): 1 score,piece blooding and big erythema (=1 mm): 3 score and erosion and ulcer:5 score.6 Observation indices6.1 Observation: the daily general clinical symptoms in mice wereobserved from beginning to end of the experiment in mice and the stool,mental status and death status were recorded.6.2 BW was recorded weekly from beginning to end of the experiment.6.3 Colon injury was observed with naked eyes to score the colon grossmorphological injury, including blood spot, piece blooding and ulcer.

7. Statistics

Data were expressed as x±S. All numerical variables were analyzed withSPSS13.0 software by one-way anava. T-test analysis was used forinter-group comparison. P<0.05 showed a statistically significantdifference.

8. Results

8.1 effect on general conditions in rats

In the model group, on 1^(st) day, formless watery stool occurred inrats, increased stool frequency and accompanied with mucus on2^(nd)˜3^(rd) days, lasting to the end of administration. In thepositive control group (420 mg/kg), on 1^(st) day, formless watery stooloccurred in rats and most of watery stool symptoms disappeared in ratson 5^(th)˜6^(th) days. In the andrographolide high-dose intragastric andintracolonical groups (90 mg/kg), on 1^(st) day, formless watery stooloccurred in rats, symptoms disappeared gradually on 2^(nd)˜3^(rd) daysand totally disappeared on 4^(th)˜5^(th) days.

8.2 Effect on UC

In the model group, the intestinal wall got thickened, ruga disappearedand small area of necrosis appeared, wide mucosal congestion, edma andulcer seen in many sites. In the positive control group, the intestinalwall was thickened mildly, a part of ruga disappeared and small area ofnecrosis appeared, mucosal congestion, edma seen in many sites and theulcer area was a little more reduced than the model group (P<0.05). Inthe andrographolide high-dose intracolonic group (90 mg/kg), symptoms ofcolon disease in rats was relieved obviously, no thickened intestinalwall found, the ruga normal, no significant mucusal congestion observedand edma and very small area of necrosis was visible topically only. Theulcer area was significantly more reduced than the model group andpositive control group (P<0.01). Compared with the andrographolidehigh-dose intragastric group, the area was reduced (P<0.05). Data wereseen in Table 4 and FIG. 2.

TABLE 4 effect of andrographolide on UC in rats by differentadministration routes Gross morphological Ulcer ratio Groups Dose(mg/kg) score (%) Model group 8.87 ± 1.71 61.87 Positive control group420 3.16 ± 1.97* 35.45* Andrographolide low-dose 30 6.71 ± 2.57 45.69*intragastric group Andrographolide high-dose 90 3.25 ± 1.98* 29.21**intragastric group Andrographolide low-dose 30 6.53 ± 2.12 38.54*intracolonic group Andrographolide high-dose 90 1.96 ± 1.09** 17.21**intracolonic group Compared with the model group: *P < 0.05: **P < 0.01

9. Conclusions

As a hapten, TNBS was mostly used in combination with ethanol to buildmodel. The mechanism of this model was set forth as follows: mucosalinjury was caused by using ethanol, TNBS, acting as hapten, infiltratedinto colon tissue to form complete antigen by combining with highpolymeric substance of tissue proteins to cause Th1 immune response andthen lead to human CD alike inflammation.

As shown in the results, the andrographolide would ameliorate thediarrhea in TNBS-induced UC model of rats and have a certain effect onimproving colon injury and ulcer ratio. Compared with theandrographolide high-dose intragastric group, the andrographolidehigh-dose intracolonic group had a stronger effect on improving coloninjury and ulcer ratio (P<0.05). It was confirmed that rectally topicaladministration would have a better effect on improving UC than theintrogastric administration, having a certain advantage of application.

Pharmacological Research 4 Therapeutical Effect on Zebra Fish Crohn'sDisease Model

1. Exploration of concentration for efficacy evaluation in zebra fishCrohn's disease model

Fish farm water of wild-type AB strain zebra fish Crohn's disease model(1 L reverse osmosis water was added with 200 mg instant sea salt withthe conductivity of 480˜510 μS/cm, pH value of 6.9˜7.2 and hardness of53.7˜71.6 mg/L CaCO₃) was added with andrographolide in theconcentration of 0 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, 100 μg/mL and500 μg/mL by volume. At each concentration, 30 zebra fish were treated,during which zebra fish death number was counted daily and the dead fishwas removed in time. After treatment, the dearth number of zebra fish ineach group was analyzed statistically to draw the optimumconcentration-effect curve by using JMP software and calculate MNLC. Byexploring concentration, solubility of the drug in DMSO was about 250mg/ml and the administration method was to dissolve the drug into thefish farm water. It was found that when concentration ≧500 ug/ml, thedrug began to precipitate and toxicity and dearth were not found inzebra fish. As a result of this, following four concentrations wereselected for efficacy evaluation in Crohn's disease in this study: 50μg/ml, 100 μg/ml, 250 μg/ml and 500 μg/ml.

2. Efficacy of andrographolide in zebra fish Crohn's disease model

According to the results of aforesaid concentration exploration, therewere 4 levels set in treatment group to evaluate the efficacy in zebrafish Crohn's disease model, respectively 50 μg/ml, 100 μg/ml, 250 μg/mland 500 μg/ml. Meanwhile, the positive control group (prednisolone infish farm water at 10 μM), blank control group and Crohn's disease modelgroup.

Crohn's disease model (mainly inflammation) was built by using TNBS and30 zebra fish were randomly divided into each treating group. 48 hoursafter treating zebra fish with the andrographolide (at aforesaid 4different concentrations), prednisolone (positive control group) andexcipient (the excipient group), 10 zebra fish were randomly taken outin each group to observe and photographed. Image analysis software wasused to analyze the pictures. Moreover, the intestine mocuscalthickness, intestinal diameter and intestinal area were observedcarefully under the microscope and the intestinal diameter andintestinal area analyzed quantitatively. Therapeutical ratio of testeddrug in zebra fish Crohn's disease was calculated in accordance with theintestinal area in each group. The calculation formula was present asfollows:

Therapeutical ratio (%)=[1−(treatment group−blank control group)/(modelgroup−blank control group)]×100%

Statistically analyzed results were expressed as mean±SD. Varianceanalysis was used for comparison among many groups and Dunnett's T testfor comparison between two groups. P<0.05 showed a statisticallysignificant difference.

As shown in FIG. 3, in the blank control group and excipient controlgroup, the intestinal mocusa was smooth, morphologically integrated,intestinal tension clear and ruga regular. In the model group (Crohn'sdisease), zebra fish bowel was dilated, the bowel area increased, theintestinal mocusa thinned and the ruga unregular, flattened or vanished.In the positive control group, Crohn's disease zebra fish bowel dilationwas reduced, the bowel area decreased, the intestinal tension and rugarecovered significantly. After being treated with low-doseandrographolide (50 ug/ml), Crohn's disease zebra fish bowel area wasreduced to a certain degree, no obvious recovery of the intestinaltension and ruga found. High-dose of andrographolide (100, 250, 500ug/ml), however, could effectively ameliorate mucosal morphology incolotis, bowel dilation reduced significantly, the bowel area close tonormal, the intestinal tension and ruga nearly recovered.

As shown in FIG. 4 and FIG. 5, after being treated with the prednisolonein the positive control group, the bowel area was reduced by (76±9.3) %,namely the therapeutical ratio of 76±9.3% in zebra fish Crohn's diseasemodel. The prednisolone (50 ug/ml) had a certain therapeutic effect onzebra fish Crohn's disease model and bowel area was reduced by(38±12.9)%. Compared with the model group, there was no statisticallysignificant difference (P>0.05). In the andrographolide high-dose group(100, 250 and 500 ug/ml), zebra fish bowel areas were reducedrespectively by (49±8.9)%, (65±14.7)% and (65±10.1)%, havingstatistically or extremely significant difference (P<0.05 and P<0.01)compared with the model group.

As shown in FIG. 6 and Table 5, a small amount of neutrophil was foundin zebra fish intestinal tissue of blank control group and excipientcontrol group. In the model group (Crohn's disease), bowel was dilatedsignificantly, a large amount of neutrophil infiltrated in bowel tissueand inflammation obvious. The prednisolone of positive drug couldsignificantly reduce neutrophil in Crohn's disease tissue. Theinflammation regression ratio was (72±4.14)%, having extremelystatistically significant difference (p<0.001) compared with the modelgroup (Crohn's disease). In the andrographolide high-dose group (100,250 and 500 ug/ml), the neutrophil infiltration in bowel tissue ofCrohn's disease zebra fish was reduced, facilitating inflammationregression. The inflammation regression ratio in bowel tissue of Crohn'sdisease were (45±3.74)%, (46±3.74)%, (63±4.42)% and (79±8.98)%, havingan extremely significant difference (p<0.001) compared with the modelgroup (Crohn's disease).

TABLE 5 fluorescence evaluation on neutrophil in Crohn's disease zebrafish model (x ± SE, n = 10) Inflammation Neutrophil Ratio of blankregression Group count control group ratio (%) Blank control group  9 ±0.53 1.00 ± 0.06 — Excipient control group 10 ± 0.27 1.07 ± 0.03 — Modelgroup 24 ± 1.17 2.70 ± 0.13 — Positive control group 14 ± 0.60* 1.53 ±0.07* 72 ± 4.1* Andrographolide 18 ± 0.54* 1.97 ± 0.06* 45 ± 3.7* (50μg/ml) Andrographolide 17 ± 0.54* 1.96 ± 0.06* 46 ± 3.7* (100 μg/ml)Andrographolide 15 ± 0.64* 1.67 ± 0.07* 63 ± 4.4* (250 μg/ml)Andrographolide 13 ± 1.30* 1.40 ± 0.15* 79 ± 9.0* (500 μg/ml) Comparedwith the model group, *P < 0.001Pharmacological Research 5 Pharmacological Study of pH-Dependent EntericTargeting Preparation

In Vitro Release Experiment

In vitro release of micropellets was determined, which were prepared bytwo formulas selected from aforesaid optimized ones. That is to say, 150mg of andrographolide enteric targeting micropellet was loaded into acapsule to measure in vitro release. Dissolution 1 method of ChinesePharmacopeia was used at rotating speed of 100 rpm with different pHsalt solution (1000 ml) as release medium. According to requirement ofChinese Pharmacopeia, after sampling, HPLC was used to measure releaseamount of drug during different period of time. The results were seen inFIG. 7 and FIG. 8. Wherein, #1 formula had the fastest release rate and#7 formula slowest. The other formulas stayed somewhere in between.

EXAMPLE 1. Preparative Example for Andrographolide Enteric-Coated Tabletand Capsule Example 1-1 Preparation of Enteric-Coated Tablet

Extraction of andrographolide: the leaves of Andrographis paniculata wassoaked in 95% (v/v) ethanol and the resulting ethanol liquid wasdecolored with activated carbon and the ethanol is recovered bydistillation to give a concentrated liquid. The liquid was allowed tostand still to have coarse crystal. Said coarse crystal was added with15 times (15×) 95% (v/v) ethanol, dissolved by heating, decolored withactivated carbon and filtered immediately. The resultant liquid wasallowed to stand still to give a light-yellow crystal byrecrystallization. The obtained crystal is refined by washing withdistilled water, chloroform and methol to have the final product ofandrographolide.

Appropriate amount of excipient was added into afore-obtainedandrographolide to prepare the enteric-coated tablet by a conventionalmethod.

The extracting method was the same as the EXAMPLE 1-1.

Appropriate amount of excipient was added into afore-obtainedandrographolide to prepare the enteric-coated capsule by a conventionalmethod.

Example 1-3 Preparation of Granule

andrographolide 100 g  microcrystalline cellulose 50 g lactose 50 gstarch 50 g surcose 250 g  to prepare 500 g granule

Method:

The extracting method was the same as the EXAMPLE 1-1. In addition,andrographolide and other excipients were screened with 100-mesh sifter,mixed well to prepare into the soft material by using appropriate amountof water, granulated with 14-mesh and sorted out.

Example 1-4 Preparation of Intestinal Suppository

The materials were mixed well according to the formula of EXAMPLE 1-3,into which the matrix was added to prepare the intestinal suppository bya conventional method.

2. Examples and Preparative Examples of Andrographolide pH-DependentEnteric Targeting Micropellet

It should be noted that the percentage of examples preparative examplesreferred to percentage by weight.

Example 2-1

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer was composed of following formula (g):

anti- Blank Eurdragit sticking pellet Andrographolide S100 plasticizeragent Surfactant 200 50 15 2.1 4.5 1.00

Wherein, said blank pellet was a blank sucrose pellet with a diameter of600 μm; said plasticizer was the triethyl citrate; said anti-stickingagent was the talc and said surfactant was the SDS (sodium dodecylsulfate);

The enteric coating layer included the Eudragit L100-55, plasticizer andanti-sticking agent and they were selected as depicted in the druglayer. The amount of plasticizer was 15 wt % of the Eudragit L100-55,the anti-sticking agent 30 wt % and weight gain of coating 5%.

Example 2-2

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer was composed of following formula (g):

anti- Blank Eurdragit sticking pellet Andrographolide S100 plasticizeragent Surfactant 200 66 20 3 6 1.32

Wherein, said blank pellet was a blank sucrose pellet with a diameter of200 μm; said plasticizer was the dibutyl sebacate; said anti-stickingagent was the glyceryl monostearate and said surfactant was theTween-80;

The enteric coating layer included the Eudragit L100-55, plasticizer andanti-sticking agent and they were selected as depicted in the druglayer. The amount of plasticizer was 15 wt % of the Eudragit L100-55,the anti-sticking agent 30 wt % and weight gain of coating 30%.

Example 2-3

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer was composed of following formula (g):

anti- Blank Eurdragit sticking pellet Andrographolide S100 plasticizeragent Surfactant 200 66 22 3.3 6.6 1.32

Wherein, said blank pellet was a blank sucrose pellet with a diameter of400 μm; said plasticizer was the propanediol; said anti-sticking agentwas the talc and said surfactant was the SDS;

The enteric coating layer included the Eudragit L100-55, plasticizer andanti-sticking agent and they were selected as depicted in the druglayer. The amount of plasticizer was 15 wt % of the Eudragit L100-55,the anti-sticking agent 30 wt % and weight gain of coating 8%.

Example 2-4

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer was composed of following formula (g):

anti- blank Eurdragit sticking pellet andrographolide L30D-55plasticizer agent surfactant 200 44 13 2 3 0

Wherein, said blank pellet was a blank sucrose pellet with a diameter of500 μm; said plasticizer was the propanediol and said anti-stickingagent was the talc;

The enteric coating layer included the Eudragit L30D-55, plasticizer andanti-sticking agent and they were selected as depicted in the druglayer. The amount of plasticizer was 15 wt % of the Eudragit L30D-55,the anti-sticking agent 30 wt % and weight gain of coating 20%.

Example 2-5

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer was composed of following formula (g):

anti- blank Eurdragit sticking pellet andrographolide S100 plasticizeragent surfactant 200 58 18 3.6 5.4 1.16

Wherein, said blank pellet was a blank sucrose pellet with a diameter of500 μm; said plasticizer was the PEG and said anti-sticking agent wasthe talc;

The enteric coating layer included the Eudragit L100-55, plasticizer andanti-sticking agent and they were selected as depicted in the druglayer. The amount of plasticizer was 15 wt % of the Eudragit L100-55,the anti-sticking agent 30 wt % and weight gain of coating 28%.

Example 2-6

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer was composed of following formula (g):

anti- blank Eurdragit sticking pellet andrographolide S100 plasticizeragent surfactant 200 15.2 40 6 12 0.4

Wherein, said blank pellet was a blank sucrose pellet with a diameter of500 μm; said plasticizer was the PEG and said anti-sticking agent wasthe talc;

The enteric coating layer included the Eudragit L100-55, plasticizer andanti-sticking agent and they were selected as depicted in the druglayer. The amount of plasticizer was 15 wt % of the Eudragit L100-55,the anti-sticking agent 30 wt % and weight gain of coating 15%.

Example 2-7

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer was composed of following formula (g):

anti- blank Eurdragit sticking pellet andrographolide S100 plasticizeragent surfactant 200 52 74 13.5 27 1.26

Wherein, said blank pellet was a blank sucrose pellet with a diameter of500 m; said plasticizer was the triethyl citrate and said anti-stickingagent was the talc;

The enteric coating layer included the Eudragit L100-55, plasticizer andanti-sticking agent and they were selected as depicted in the druglayer. The amount of plasticizer was 15 wt % of the Eudragit L100-55,the anti-sticking agent 30 wt % and weight gain of coating 15%.

Example 2-8

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer was composed of following formula (g):

anti- blank Eurdragit sticking pellet andrographolide S100 plasticizeragent surfactant 200 66 20 3 6 1.32

Wherein, said blank pellet was a blank sucrose pellet with a diameter of600 μm; said plasticizer was the triethyl citrate; said anti-stickingagent was the talc and said surfactant was the SDS;

The enteric coating layer included the Eudragit L100-55, plasticizer andanti-sticking agent and they were selected as depicted in the druglayer. The amount of plasticizer was 15 wt % of the Eudragit L100-55,the anti-sticking agent 30 wt % and weight gain of coating 8%.

Example 2-9

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer contained the andrographolide, the polymer A dissolved undercondition of pH≧7.0: said ratio of the andrographolide and polymer A is1:2 by weight; weight gain of the drug layer was 20 wt %;

Said enteric coating layer contained the polymer B dissolved undercondition of pH≧5.5 and weight gain of the enteric coating layer was 8wt %. The proportion of plasticizer and anti-sticking agent was the samewith the one of polymer A and plasticizer and anti-sticking agent in thedrug layer.

Wherein, said polymer A was the copolymer of methacrylic acid and methylmethacrylate and polymer B is the copolymer of methacrylic acid andethyl acrylate.

Aforesaid plasticizer was the triethyl citrate and anti-sticking agentthe talc.

Example 2-10

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer contained the andrographolide, the polymer A dissolved undercondition of pH≧7.0; said ratio of the andrographolide and polymer A is1:0.2 by weight; weight gain of the drug layer was 100 wt %; saidenteric coating layer contained the polymer B dissolved under conditionof pH≧5.5 and weight gain of the enteric coating layer was 20 wt %.

Said polymer A was the copolymer of methacrylic acid and methylmethacrylate in a ratio of 1:2 and polymer B was the copolymer ofmethacrylic acid and ethyl acrylate in a ratio of 1:1.

Aforesaid plasticizer was the dibutyl sebacate and anti-sticking agentthe glyceryl monostcarate.

Example 2-11

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer contained the andrographolide, the polymer A dissolved undercondition of pH≧7.0; said ratio of the andrographolide and polymer A is1:1.5 by weight; weight gain of the drug layer was 30 wt %; said entericcoating layer contained the polymer B dissolved under condition ofpH≧5.5 and weight gain of the enteric coating layer was 10 wt %. Theproportion of plasticizer and anti-sticking agent was the same with theone of polymer A and plasticizer and anti-sticking agent in the druglayer.

Said polymer A was the copolymer of methacrylic acid and methylmethacrylate in a ratio of 1:2 and polymer B was the copolymer ofmethacrylic acid and ethyl acrylate in a ratio of 1:1.

Example 2-12

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer contained the andrographolide, the polymer A dissolved undercondition of pH≧7.0, the plasticizer, anti-sticking agent, pigment,hydrophilic polymer and surfactant; said ratio of the andrographolideand polymer A is 1:2 by weight; weight gain of the drug layer was 100 wt%; said plasticizer was selected from the triethyl citrate, accountingfor 10 wt % of the polymer A; said anti-sticking agent was selected fromthe talc, accounting for 25 wt % of the polymer A.

Said enteric coating layer contained the polymer B dissolved undercondition of pH≧5.5 and weight gain of the enteric coating layer was 20wt %. The proportion of plasticizer and anti-sticking agent was the samewith the one of polymer A and plasticizer and anti-sticking agent in thedrug layer.

Said polymer A was the copolymer of methacrylic acid and methylmethacrylate in a ratio of 1:2 and polymer B was the copolymer ofmethacrylic acid and ethyl acrylate in a ratio of 1:1.

Aforesaid plasticizer was the triethyl citrate and anti-sticking agentthe talc.

Example 2-13

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer contained the andrographolide, the polymer A dissolved undercondition of pH≧7.0, the plasticizer, anti-sticking agent, pigment,hydrophilic polymer and surfactant; said ratio of the andrographolideand polymer A is 1:0.5 by weight; weight gain of the drug layer was 80wt %; said plasticizer was selected from the dibutyl sebacate,accounting for 70 wt % of the polymer A; said anti-sticking agent wasselected from the talc, accounting for 100 wt % of the polymer A.

Said enteric coating layer contained the polymer B dissolved undercondition of pH≧5.5 and weight gain of the enteric coating layer was 18wt %. The proportion of plasticizer and anti-sticking agent was the samewith the one of polymer A and plasticizer and anti-sticking agent in thedrug layer.

Said polymer A was the copolymer of methacrylic acid and methylmethacrylate in a ratio of 1:2 and polymer B was the copolymer ofmethacrylic acid and ethyl acrylate in a ratio of 1:1.

Aforesaid plasticizer was the triethyl citrate and anti-sticking agentthe talc.

Example 2-14

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer contained the andrographolide, the polymer A dissolved undercondition of pH≧7.0, the plasticizer, anti-sticking agent, pigment,hydrophilic polymer and surfactant; said ratio of the andrographolideand polymer A is 1:1 by weight: weight gain of the drug layer was 50 wt%; said plasticizer was selected from the propanediol, accounting for 20wt % of the polymer A; said anti-sticking agent was selected from thetalc, accounting for 30 wt % of the polymer A.

Said enteric coating layer contained the polymer B dissolved undercondition of pH≧5.5 and weight gain of the enteric coating layer was 15wt %. The proportion of plasticizer and anti-sticking agent was the samewith the one of polymer A and plasticizer and anti-sticking agent in thedrug layer.

Said polymer A was the copolymer of methacrylic acid and methylmethacrylate in a ratio of 1:2 and polymer B was the copolymer ofmethacrylic acid and ethyl acrylate in a ratio of 1:1.

Aforesaid plasticizer was the triethyl citrate and anti-sticking agentthe talc.

Example 2-15

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer contained the andrographolide, the polymer A dissolved undercondition of pH≧7.0, the plasticizer, anti-sticking agent, pigment,hydrophilic polymer and surfactant; said ratio of the andrographolideand polymer A is 1:1.5 by weight; weight gain of the drug layer was 60wt %; said plasticizer was selected from the PEG, accounting for 50 wt %of the polymer A; said anti-sticking agent was selected from the talc,accounting for 80 wt % of the polymer A.

Said enteric coating layer contained the polymer B dissolved undercondition of pH≧5.5 and weight gain of the enteric coating layer was 16wt %. The proportion of plasticizer and anti-sticking agent was the samewith the one of polymer A and plasticizer and anti-sticking agent in thedrug layer.

Said polymer A was the copolymer of methacrylic acid and methylmethacrylate in a ratio of 1:2 and polymer B was the copolymer ofmethacrylic acid and ethyl acrylate in a ratio of 1:1.

Aforesaid plasticizer was the triethyl citrate and anti-sticking agentthe talc.

Example 2-16

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer contained the andrographolide, the polymer A dissolved undercondition of pH≧7.0, the plasticizer, anti-sticking agent, pigment,hydrophilic polymer and surfactant; said ratio of the andrographolideand polymer A is 1:1.5 by weight; weight gain of the drug layer was 60wt %; said plasticizer was selected from the PEG, accounting for 50 wt %of the polymer A; said anti-sticking agent was selected from theglyceryl monostearate, accounting for 20 wt % of the polymer A.

Said enteric coating layer contained the polymer B dissolved undercondition of pH≧5.5 and weight gain of the enteric coating layer was 16wt %. The proportion of plasticizer and anti-sticking agent was the samewith the one of polymer A and plasticizer and anti-sticking agent in thedrug layer.

Said polymer A was the copolymer of methacrylic acid and methylmethacrylate in a ratio of 1:2 and polymer B was the copolymer ofmethacrylic acid and ethyl acrylate in a ratio of 1:1.

Aforesaid plasticizer was the triethyl citrate and anti-sticking agentthe talc.

Example 2-17

Andrographolide enteric targeting micropellet was composed of a blankpellet, a drug layer and an enteric coating layer, wherein said druglayer contained the andrographolide, the polymer A dissolved undercondition of pH≧7.0, the plasticizer, anti-sticking agent, pigment,hydrophilic polymer and surfactant; said ratio of the andrographolideand polymer A is 1:1.5 by weight; weight gain of the drug layer was 60wt %; said plasticizer was selected from the PEG, accounting for 50 wt %of the polymer A; said anti-sticking agent was selected from theglyceryl monostearate, accounting for 20 wt % of the polymer A.

Said enteric coating layer contained the polymer B dissolved undercondition of pH≧5.5 and weight gain of the enteric coating layer was 16wt %. The proportion of plasticizer and anti-sticking agent was the samewith the one of polymer A and plasticizer and anti-sticking agent in thedrug layer.

Said polymer A was the copolymer of methacrylic acid and methylmethacrylate in a ratio of 1:2 and polymer B was the copolymer ofmethacrylic acid and ethyl acrylate in a ratio of 1:1.

Aforesaid plasticizer was the triethyl citrate and anti-sticking agentthe talc.

Micropellets in aforesaid Examples were prepared by following method.

Preparative Example 2-1

(1) applying drug to the blank pelleta). dispersing the polymer A into a pharmaceutical solvent to let themdissolve fully by mechanical stirring: adding the excipient into thepolymer A solution and then adding the andrographolide to have thepolymer A coating solution by well stirring;b). weighing the blank pellet and charging into a fluidized bed;adjusting air flow to such a degree that the micropellet was wellfluidized; opening the heating device and until temperature of thematerial reaches preset value, the peristaltic pump was started to makethe polymer A coating solution atomized through a spray gun to obtain adrug-loading micropellet by dispersing on the surface of said blankpellet;(2) preparation of the enteric coating layera). dispersing the polymer B into a pharmaceutical solvent to let themdissolve fully by mechanical stirring; adding the excipient into thepolymer B solution to have the polymer B coating solution by wellstirring;b). charging aforesaid drug-loading micropellets into a bottom-spraydevice of the fluidized bed, and the polymer B coating solution isuniformly spread to form the enteric coating layer; the weight gain is 5wt %.

Preparative Example 2-2

(1) applying drug to the blank pelleta). dispersing the polymer A into a pharmaceutical solvent to let themdissolve fully by mechanical stirring; adding the excipient into thepolymer A solution and then adding the andrographolide to have thepolymer A coating solution by well stirring;b). weighing the blank pellet and charging into a fluidized bed;adjusting air flow to such a degree that the micropellet was wellfluidized; opening the heating device and until temperature of thematerial reaches preset value, the peristaltic pump was started to makethe polymer A coating solution atomized through a spray gun to obtain adrug-loading micropellet by dispersing on the surface of said blankpellet; in(2) preparation of the enteric coating layera). dispersing the polymer B into a pharmaceutical solvent to let themdissolve fully by mechanical stirring; adding the excipient into thepolymer B solution to have the polymer B coating solution by wellstirring;b). charging aforesaid drug-loading micropellets into a bottom-spraydevice of the fluidized bed, and the polymer B coating solution isuniformly spread to form the enteric coating layer, the weight gain is30 wt %.

Preparative Example 2-3

a). dispersing the polymer A into a pharmaceutical ethanol to make thecontent of polymer A at 5 wt %; fully dissolving by high-speed shearingmechanical stirring and continuing to stir uniformly; adding theexcipient of the plasticizer, anti-sticking agent and the surfactant ofsodium dodecyl sulfate into the polymer A solution and then adding theandrographolide to have the polymer A coating solution by well stirring;b). weighing the blank sucrose pellet in a diameter of 200 μm andcharging into a fluidized bed; adjusting air flow to such a degree thatthe micropellet is well fluidized; opening the heating device to keepthe temperature of the material at 25° C. and until the temperaturereaches preset value, the peristaltic pump is started to make thepolymer A coating solution atomized through a spray gun to obtain adrug-loading micropellet by dispersing on the surface of said blankpellet;(2) preparation of the enteric coating layera). dispersing the polymer B into the pharmaceutical ethanol to let themdissolve fully by high-speed shearing mechanical stirring; adding theexcipient of the plasticizer and anti-sticking agent into the polymer Bsolution to have the polymer B coating solution by well stirring;b). charging aforesaid drug-loading micropellets into a bottom-spraydevice of the fluidized bed, and the polymer B coating solution isuniformly spread to form the enteric coating layer, the weight gain is 8wt %.

Preparative Example 2-4

(1) applying drug to the blank pelleta). dispersing the polymer A into a pharmaceutical ethanol to make thecontent of polymer A at 5 wt %; fully dissolving by high-speed shearingmechanical stirring and continuing to stir uniformly; adding theexcipient of the plasticizer, anti-sticking agent and the surfactant ofsodium dodecyl sulfate into the polymer A solution and then adding theandrographolide to have the polymer A coating solution by well stirring;b). weighing the blank sucrose pellet in a diameter of 600 μm andcharging into a fluidized bed; adjusting air flow to such a degree thatthe micropellet is well fluidized; opening the heating device to keepthe temperature of the material at 35° C. and until the temperaturereaches preset value, the peristaltic pump is started to make thepolymer A coating solution atomized through a spray gun to obtain adrug-loading micropellet by dispersing on the surface of said blankpellet;(2) preparation of the enteric coating layera). dispersing the polymer B into the pharmaceutical ethanol to let themdissolve fully by high-speed shearing mechanical stirring; adding theexcipient of the plasticizer and anti-sticking agent into the polymer Bsolution to have the polymer B coating solution by well stirring;b). charging aforesaid drug-loading micropellets into a bottom-spraydevice of the fluidized bed, and the polymer B coating solution isuniformly spread to form the enteric coating layer the weight gain is 20wt %.

Preparative Example 2-5

(1) applying drug to the blank pelleta). dispersing the polymer A into a pharmaceutical ethanol to make thecontent of polymer A at 5 wt %; fully dissolving by high-speed shearingmechanical stirring and continuing to stir uniformly; adding theexcipient of the plasticizer, anti-sticking agent and the surfactant ofsodium dodecyl sulfate into the polymer A solution and then adding theandrographolide to have the polymer A coating solution by well stirring;b). weighing the blank sucrose pellet in a diameter of 400 μm andcharging into a fluidized bed; adjusting air flow to such a degree thatthe micropellet is well fluidized; opening the heating device to keepthe temperature of the material at 32° C. and until the temperaturereaches preset value, the peristaltic pump is started to make thepolymer A coating solution atomized through a spray gun to obtain adrug-loading micropellet by dispersing on the surface of said blankpellet;(2) preparation of the enteric coating layera). dispersing the polymer B into the pharmaceutical ethanol to let themdissolve fully by high-speed shearing mechanical stirring; adding theexcipient of the plasticizer and anti-sticking agent into the polymer Bsolution to have the polymer B coating solution by well stirring;b). charging aforesaid drug-loading micropellets into a bottom-spraydevice of the fluidized bed, and the polymer B coating solution isuniformly spread to form the enteric coating layer; the weight gain is15 wt %.

Preparative Example 2-6

Micropellets obtained from Example 2-1˜2-17 were prepared to have theconventional granule and capsule.

What is claimed is:
 1. A method of preparing an andrographolide entericmicropellet of composed of a blank pellet, a drug layer and an entericcoating layer, wherein said drug layer contains andrographolide, apolymer A that dissolves under a condition of pH≧7.0 and an excipient;where said ratio of the andrographolide and polymer A is 1:2˜1:0.2 byweight; and wherein the weight gain of the drug layer is 20 wt %˜100 wt%; said enteric coating layer containing a polymer B that dissolvesunder a condition of pH≧5.5 and an excipient; and wherein the weightgain of the enteric coating layer is 5 wt %˜30 wt %, comprising thefollowing steps: (1) applying andrographolide to a blank micropellet bya). dispersing the polymer A into a pharmaceutical solvent to allow thepolymer A to dissolve in the solvent fully by mechanical stirring toobtain a polymer A solution: adding an excipient into the polymer Asolution and then adding andrographolide to the polymer A solution andthen stirring well to provide a polymer A coating solution; b). weighingthe blank micropellet and charging the blank micropellet into afluidized bed; adjusting the air flow in the fluidized bed to such adegree that the micropellet is well fluidized; starting a heating deviceand heating the well fluidized blank micropellet until temperature ofthe well fluidized blank micropellet material reaches a preset value;starting a peristaltic pump which atomizes the polymer A coatingsolution through a spray gun to uniformly disperse the polymer A coatingsolution on the surface of the blank micropellet to obtain a drug-loadedmicropellet; (2) preparing the enteric coating layer by a). dispersingthe polymer B into a pharmaceutical solvent to allow the polymer B todissolve in the solvent fully by mechanical stirring to obtain a polymerB solution; adding an excipient into the polymer B solution and thenstirring well to provide a polymer B coating solution; b). chargingaforesaid andrographolide-loaded micropellet into a bottom-spray deviceof a fluidized bed, and uniformly spreading the polymer B coatingsolution to form the enteric coating layer; wherein the weight gain ofthe enteric coating layer is 5 wt %˜30 wt %.
 2. The preparation methodaccording to claim 8 comprising following steps: (1) applying drug tothe blank micropellet by a). dispersing the polymer A into thepharmaceutical solvent which is ethanol such that the content of polymerA is 5 wt %; fully dissolving the polymer A in the ethanol by mechanicalstirring to form the polymer A solution and continuing to uniformly stirthe polymer A solution; adding excipient which is plasticizer,anti-sticking agent and the surfactant sodium dodecyl sulfate into thepolymer A solution, and then adding the andrographolide to the polymer Asolution and then stirring well to provide the polymer A coatingsolution; b). weighing the blank micropellet which is a sucrosemicropellet having a diameter of 200˜600 μm and charging the sucrosemicropellet into a fluidized bed; adjusting air flow to such a degreethat the sucrose micropellet is well fluidized; starting the heatingdevice and heating the well fluidized sucrose micropellet until thetemperature reaches the preset value of 25˜35° C. starting theperistaltic pump which atomizes the polymer A coating solution through aspray gun to uniformly disperse the polymer A coating solution on thesurface of the sucrose micropellet to obtain a drug-loaded sucrosemicropellet; (2) preparing the enteric coating layer by a). dispersingthe polymer B into the pharmaceutical solvent ethanol to allow thepolymer B to dissolve in the ethanol fully by high-speed shearingmechanical stirring to obtain a polymer B solution; adding the excipientof the plasticizer and anti-sticking agent into the polymer B solutionand stirring well to provide the polymer B coating solution; b).charging aforesaid drug-loaded sucrose micropellet into a bottom-spraydevice of the fluidized bed, and uniformly spreading the polymer Bcoating solution to form the enteric coating layer; wherein the weightgain of the enteric coating layer is 8 wt %˜20 wt %.
 3. The method ofclaim 1 wherein the ratio of the andrographolide and polymer A is1:2.5˜1:0.5 by weight.
 4. The method of claim 1 wherein the excipient ofthe drug layer is selected from the group consisting of plasticizer,antisticking agent, pigment, hydrophilic polymer and surfactant andwherein the excipient of the enteric coating layer is selected from thegroup consisting of plasticizer and antisticking agent.
 5. The method ofclaim 4 wherein the ingredients are present in proportion by weightparts: blank micropellet:andrographolide:polymerA:plasticizer:antisticking agent:surfactant=200:(10-100):(10-100):(1-15):(1-30):(0-3).
 6. The method of claim 4 whereinthe ingredients are present in proportion by weight parts: blankmicropellet:andrographolide:polymer A:plasticizer:antistickingagent:surfactant=200:(15-66):(13-74):(2-13.5):(3-27):(0-1.32).
 7. Themethod of claim 4 wherein the ingredients are present in proportion byweight parts: blank micropellet:andrographolide:polymerA:plasticizer:antisticking agent:surfactant=200:(20-50):(30-60):(5-10):(5-20):(0.5-1.2).